The invention relates to the identification of agents that can modulate the taste response in humans (in particular sweetness enhancers) through assays based on novel sweet receptor proteins, heterologous expression systems containing nucleic acid constructs forming said sweet receptor protein, and the use of the sweet receptor protein in screening. The sweetness enhancers render certain foods more palatable or increase patient compliance in oral pharmaceutics and nutraceutics. Such sweetness enhancers include sweet tastants that elicit a taste response in humans.
Sweetness modulators and in particular sweetness enhancers are of great use and interest to the food and flavor industry, for example, to allow reduction of the level of sweeteners, including sugars and artificial sweeteners, in consumable products. The use of sweetness enhancers can reduce calories, prevent teeth from damage by sugars, and avoid or reduce the bitter/metallic off- and aftertastes associated with many artificial sweeteners.
The detection of sweet taste is known to be mediated by a receptor, TAS1R, comprised of two subunits, T1R2 and T1R3, which are specifically expressed in taste receptor cells, and form a dimeric sweet taste receptor complex (T1R2/T1R3 heterodimer). Both subunits belong to the family of so-called “G-protein coupled receptors” or GPCRs, in particular class-C GPCRs.
Like most other GPCRs, the class-C receptors have a heptahelical transmembrane domain (TMD). However, unlike other types of GPCRs, the class-C GPCRs also have a large extracellular domain composed of two parts: a “venus flytrap module” (VFTM) that is involved in ligand binding; and a cysteine-rich domain (CRD) that contains nine highly conserved cysteines and that links the VFTM to the TMD. A variable length intracellular C-terminal tail completes the class-C receptor.
Activation of the sweet receptor response was thought to require both subunits of the dimeric sweet receptor complex, and to date, all sweeteners tested activate the T1R2/T1R3 heterodimer. Published tests conducted with the separate subunits of the human sweet receptor (T1R2 homomer or T1R3 homomer) have shown no activity, while the T1R2/T1R3 heterodimer responds to a broad spectrum of chemically diverse sweeteners, ranging from natural sugars (sucrose, fructose, glucose, maltose), sweet amino acids (D-tryptophan), and artificial sweeteners (acesulfame-K, aspartame, cyclamate, saccharin, sucralose), to sweet tasting proteins (monellin, thaumatin, brazzein) (see for example Li et al., 2002, Proc. Natl. Acad. Sci. USA, 99(7), 4692-6).
Currently known screens for sweetness modulators employ the T1R2/T1R3 heterodimeric sweet receptor. These screens usually identify or characterize a sweetness modulator by comparing the results of samples with and without a potential modulator in the presence of a sweetener. However, sweeteners and in particular sugars have a great effect on osmolarity, and/or are viscous. Due to changes in properties of the samples such as viscosity and osmolarity, artifacts may occur that cause incorrect results when using standard screening methods.
Another disadvantage of known screens is that the wildtype T1R2/T1R3 receptor comprises several binding domains, in particular the extracellular amino terminal domains including the venus flytrap (“VFT”) domain that bind to carbohydrate sweeteners such as sucrose, glucose, fructose as well as the artificial sweeteners aspartame and sucralose. Therefore, a screen for specific modulators of specific ligands, and in particular for ligands of the transmembrane domains (“TMD(s)”), excluding the VFT ligands, is not possible with known screening methods.
In order to prevent identification of agents that may compete with sugars for binding to the receptor, a screen that allows identification of putative sweet receptor enhancers that bind at a site physically distinct from the VFT domains, and in particular in the TMD and/or cysteine-rich domains, would be desirable. The present invention addresses this need. For example, the inventive method using chimeric T1R receptors created by the applicant (CSR:T1R2 or CSR:T1R3 chimeric receptors, collectively the CSR:T1R chimeric receptors (“CSR” refers to the calcium-sensing receptor)) indicates that the sweet compound cyclamate binds in the TMD of T1R3, thereby activating the heterodimeric T1R2/T1R3 receptor complex.
To date, it was unknown that the TMD of one of the TAS1R monomers could bind sweet compounds and additionally activate G-proteins in the absence of the other obligate monomeric partner. Previously, the field believed the presence of both subunits was essential for signal transduction. Applicant has found that a novel receptor protein (“T1R2-TMD”) corresponding to a heavily truncated sequence of the T1R2 homomer of the T1R2/T1R3 heterodimer receptor complex forms, surprisingly, a functional sweet receptor that binds to a sweet ligand and is able to activate G-proteins. The novel receptor protein T1R2-TMD was found to have a different agonist spectrum than the full-length T1R2 homomer. The former was surprisingly found to be able not only to bind to ligands but also to activate downstream signaling.
The methods provided herein permit the identification of ligands that bind to and/or activate the transmembrane and intra-cellular domains of T1R2 and/or T1R3. Accordingly, cells expressing either CSR:T1R chimeric receptors or T1R2-TMD and a G-protein, are contacted with test agents, optionally in combination with known or newly determined sweet tastants, to determine the properties of said agents as sweetness enhancers. The assays provided herein may therefore be used to identify a tested agent as sweet tastant or enhancer of the sweet response. The functional effects of the agent on the receptor and G-protein are determined by a suitable functional assay, for example, an assay that measures changes in parameters of the transduction pathways such as intracellular IP3 and Ca2+, or by other G-protein specific assays such as labeling with GTPγS, according to techniques known in the art. Alternatively, binding assays may be used to determine ligand binding to CSR:T1R chimeric receptors or T1R2-TMD. The identified agent can then be tested for its activity as a sweetness enhancer, according to techniques known in the art, described without limitation herein below.
In practicing the various aspects and embodiments described herein in relation to cloning, elucidating ligand-receptor pairs, and finding enhancers of the sweet response, recourse is made to conventional techniques in molecular biology, microbiology and recombinant technology and sweetness testing. These include the various known methods suitable for G-protein coupled receptors (GPCRs) including CSR:T1R chimeric receptors or T1R2-TMD. Accordingly, the skilled person is fully apprised of such techniques and as such they are hereafter treated only summarily in order to more fully describe the context of the present invention.